Article

Substrate-induced transmembrane signaling in the cobalamin transporter BtuB

Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908, USA.
Nature Structural Biology 06/2003; 10(5):394-401. DOI: 10.1038/nsb914
Source: PubMed

ABSTRACT

The outer membranes of Gram-negative bacteria possess transport proteins essential for uptake of scarce nutrients. In TonB-dependent transporters, a conserved sequence of seven residues, the Ton box, faces the periplasm and interacts with the inner membrane TonB protein to energize an active transport cycle. A critical mechanistic step is the structural change in the Ton box of the transporter upon substrate binding; this essential transmembrane signaling event increases the affinity of the transporter for TonB and enables active transport to proceed. We have solved crystal structures of BtuB, the outer membrane cobalamin transporter from Escherichia coli, in the absence and presence of cyanocobalamin (vitamin B(12)). In these structures, the Ton box is ordered and undergoes a conformational change in the presence of bound substrate. Calcium has been implicated as a necessary factor for the high-affinity binding (K(d) approximately 0.3 nM) of cyanocobalamin to BtuB. We observe two bound calcium ions that order three extracellular loops of BtuB, thus providing a direct (and unusual) structural role for calcium.

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    • "One is the nucleotide-bound intermediate state (protein data bank (PDB) code: 4FI3) and the other is the apo BtuCD–BtuF complex (PDB code: 4DBL), as shown in Figure 1a,b, respectively. BtuF is the substrate binding protein that binds vitamin B12 with high affinity and specificity in the bacterial periplasm, and then delivers it to the transporter[15,16]. BtuCD is the ABC transporter that mediates the uptake of the substrate into the cell, which consists of two transmembrane domains (TMDs) (i.e., BtuC subunits) and two cytoplasmic nucleotide-binding domains (NBDs) (i.e., BtuD subunits), as shown in Figure 1a. "
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    ABSTRACT: BtuCD-BtuF from Escherichia coli is a binding protein-dependent adenosine triphosphate (ATP)-binding cassette (ABC) transporter system that uses the energy of ATP hydrolysis to transmit vitamin B12 across cellular membranes. Experimental studies have showed that during the transport cycle, the transporter undergoes conformational transitions between the "inward-facing" and "outward-facing" states, which results in the open-closed motions of the cytoplasmic gate of the transport channel. The opening-closing of the channel gate play critical roles for the function of the transporter, which enables the substrate vitamin B12 to be translocated into the cell. In the present work, the extent of opening of the cytoplasmic gate was chosen as a function-related internal coordinate. Then the mean-square fluctuation of the internal coordinate, as well as the cross-correlation between the displacement of the internal coordinate and the movement of each residue in the protein, were calculated based on the normal mode analysis of the elastic network model to analyze the function-related motions encoded in the structure of the system. In addition, the key residues important for the functional motions of the transporter were predicted by using a perturbation method. In order to facilitate the calculations, the internal coordinate was introduced as one of the axes of the coordinate space and the conventional Cartesian coordinate space was transformed into the internal/Cartesian space with linear approximation. All the calculations were carried out in this internal/Cartesian space. Our method can successfully identify the functional motions and key residues for the transporter BtuCD-BtuF, which are well consistent with the experimental observations.
    Full-text · Article · Aug 2015 · International Journal of Molecular Sciences
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    • "The TonB-dependent vitamin B 12 importer BtuCED (TCID 3.A.1.13) has been identified in E. coli (de Veaux et al. 1986) and imports vitamin B 12 in corrinoids (Borths et al. 2002; Chimento et al. 2003; Locher et al. 2002) to the periplasmic binding protein BtuF (Hvorup et al. 2007). However, the requirement of a vitamin B 12 ABC permease for full virulence has not yet been established in an in vivo infection model. "
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    ABSTRACT: The ATP-binding cassette transporter superfamily is present in all three domains of life. This ubiquitous class of integral membrane proteins have diverse biological functions, but their fundamental role involves the unidirectional translocation of compounds across cellular membranes in an ATP coupled process. The importance of this class of proteins in eukaryotic systems is well established as typified by their association with genetic diseases and roles in the multi-drug resistance of cancer. In stark contrast, the ABC transporters of prokaryotes have not been exhaustively investigated due to the sheer number of different roles and organisms in which they function. In this review, we examine the breadth of functions associated with microbial ABC transporters in the context of their contribution to bacterial pathogenicity and virulence.
    Full-text · Article · Jan 2012 · Protoplasma
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    • "). These proteins have been analyzed in great detail and the crystal structures of BtuB (Chimento et al., 2003), the SBP BtuF (Borths et al., 2002), the complex of BtuC (TMD) and BtuD (NBD) (Locher et al., 2002) and the BtuCD-BtuF (Hvorup et al., 2007) holotransporter have been determined. Related ABC transporters are widespread among bacteria and archaea, and the respective genes are regulated by adenosylcobalamin-dependent riboswitch (B12 element) in most cases (Rodionov et al., 2003). "
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    ABSTRACT: Since their discovery in the 1960s as 'osmotic shock-sensitive' transporters, a plethora of so-called binding protein-dependent (canonical) ATP-binding cassette (ABC) importers has been identified in bacteria and archaea. Their cellular functions go far beyond the uptake of nutrients. Canonical ABC importers play important roles in the maintenance of cell integrity, responses to environmental stresses, cell-to-cell communication and cell differentiation and in pathogenicity. A new class of abundant micronutrient importers, the 'energy-coupling factor' (ECF) transporters, was originally identified by functional genomics. ABC ATPases are an integral part of both canonical ABC and ECF importers. Fundamental differences include the modular architecture and the independence of ECF systems of extracytoplasmic solute-binding proteins. This review describes the roles of both types of transporters in diverse physiological processes including pathogenesis, points to the differences in modular assembly and depicts their common traits.
    Preview · Article · Apr 2010 · FEMS microbiology reviews
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